In the parent grant application the role of regulation of alpha- ketoglutarate (alphaKG) uptake in the homeostatic control of glutamatergic neurotransmission is reviewed. Investigations on the significance of this uptake in vivo is the focus of that grant. In contrast, here we propose to investigate the mechanism by which alphaKG uptake is regulated by glutamate, glutamine, and a novel protein, transport activating factor (TAF). Each one of these modulators exerts a homeostatic control at the membrane level, and their mechanism should extend to other transporters of neurotransmitters or their transporters. The strategy we will use is to hypotonically lyse synaptosomes, and reseal them with a defined intracellular medium. This will allow us to determine i) ionic requirements for transport, and for modulation of transport, of alphaKG; ii) whether intracellular components (e.g., protein kinases, endoplasmic reticulum, mitochondria) are required for modulation of transport of alphaKG; and iii) to test the effect of specific protein kinases on alphaKG transporter properties. The glutamate transporter will be studied in parallel, for its functional relevance, as well as for technical advantages (it is simpler and better understood). The hypothesis proposed, and to be tested, is that: i) The alphaKG transporter requires co-transport of Na+ (inward) and K+ (outward), and is chloride independent. ii) Modulation of the alphaKG transporter by glutamate and by TAF require intracellular components (a pyridoxal phosphate-dependent enzyme and the endoplasmic reticulum, respectively). The latter mechanism is analogous to insulin regulation of glucose transport. iii) Phosphorylation of internal (but not external) sites of the alphaKG transporter will decrease its velocity and/or increase the allosteric modulation by glutamate.